Table_2_Absolute and relative quantitation of amylase/trypsin-inhibitors by LC-MS/MS from wheat lines obtained by CRISPR-Cas9 and RNAi.xlsx

Quantitation of wheat proteins is still a challenge, especially regarding amylase/trypsin-inhibitors (ATIs). A selection of ATIs was silenced in the common wheat cultivar Bobwhite and durum wheat cultivar Svevo by RNAi and gene editing, respectively, in order to reduce the amounts of ATIs. The controls and silenced lines were analyzed after digestion to peptides by LC-MS/MS with different approaches to evaluate changes in composition of ATIs. First, a targeted method with stable isotope dilution assay (SIDA) using labeled peptides as internal standards was applied. Additionally, four different approaches for relative quantitation were conducted, in detail, iTRAQ labeled and label free quantitation (LFQ) combined with data dependent acquisition (DDA) and data independent acquisition (DIA). Quantitation was performed manually (Skyline and MASCOT) and with different proteomics software tools (PLGS, MaxQuant, and PEAKS X Pro). To characterize the wheat proteins on protein level, complementary techniques as high-performance liquid chromatography (HPLC) and gel electrophoresis were performed. The targeted approach with SIDA was able to quantitate all ATIs, even at low levels, but an optimized extraction is necessary. The labeled iTRAQ approach revealed an indistinct performance. LFQ with low resolution equipment (IonTrap) showed similar results for major ATIs, but low abundance ATIs as CM1, were not detectable. DDA measurements with an Orbitrap system and evaluation using MaxQuant showed that the relative quantitation was dependent on the wheat species. The combination of manual curation of the MaxQuant search with Skyline revealed a very good performance. The DIA approach with analytical flow found similar results compared to absolute quantitation except for some minor ATIs, which were not detected. Comparison of applied methods revealed that peptide selection is a crucial step for protein quantitation. Wheat proteomics faces challenges due to the high genetic complexity, the close relationship to other cereals and the incomplete, redundant protein database requiring sensitive, precise and accurate LC-MS/MS methods.

小麦蛋白质的定量分析仍然是一项挑战，尤其在针对淀粉酶/胰蛋白酶抑制剂（ATIs）方面。通过对Bobwhite普通小麦品种和Svevo硬质小麦品种分别采用RNA干扰和基因编辑技术，选择性地沉默了ATIs，以降低ATIs的含量。在通过液相色谱-串联质谱（LC-MS/MS）对不同方法进行分析后，对肽段进行了消化，以评估ATIs组成的改变。首先，采用标记肽段作为内标的目标方法，并结合稳定同位素稀释法（SIDA）进行定量。此外，还实施了四种不同的相对定量方法，具体包括iTRAQ标记和无标记定量（LFQ）结合数据依赖采集（DDA）和数据非依赖采集（DIA）。定量分析通过手动操作（Skyline和MASCOT）以及不同的蛋白质组学软件工具（PLGS、MaxQuant和PEAKS X Pro）进行。为了在蛋白质水平上表征小麦蛋白，还采用了高性能液相色谱（HPLC）和凝胶电泳等互补技术。采用SIDA的目标方法能够定量所有ATIs，即使是在低水平下，但也需要优化的提取。标记的iTRAQ方法表现出模糊的性能。低分辨率设备（IonTrap）的LFQ显示了对主要ATIs的相似结果，但对于低丰度的ATIs，如CM1，则无法检测。使用Orbitrap系统进行DDA测量，并通过MaxQuant进行评估，表明相对定量依赖于小麦品种。将MaxQuant搜索的手动编辑与Skyline相结合，揭示了出色的性能。DIA方法在分析流中找到了与绝对定量相似的结果，除了某些微量的ATIs未检测到。对应用方法的比较显示，肽段选择是蛋白质定量分析的关键步骤。由于小麦蛋白质具有高度的遗传复杂性、与其他谷物的密切关系以及不完整、冗余的蛋白质数据库，小麦蛋白质组学面临着挑战，需要敏感、精确和准确的液相色谱-串联质谱（LC-MS/MS）方法。